Morpholinos and their peptide conjugates: therapeutic promise and challenge for Duchenne muscular dystrophy.

Exon-skipping efficacies of phosphodiamidate morpholino oligomers (PMOs) or the conjugates of PMOs with cell-penetrating peptides (PPMOs) have been tested in various animal models of Duchenne muscular dystrophy (DMD), including mdx mice, utrophin-dystrophin double-knockout mice, and CXMD dogs, as well as in DMD patients in clinical trials. The studies have shown that PMOs can diffuse into leaky muscle cells, modify splicing of DMD transcripts, induce expression of partially functional dystrophin, and improve function of some skeletal muscles. PMOs are non-toxic, with a report of mdx mice tolerating a 3g/kg dose, and no drug-related safety issue in human has been reported. However, because of their poor cell uptake and rapid renal clearance, large and frequently repeated doses of PMOs are likely required for functional benefit in some skeletal muscles of DMD patients. In addition, PMOs do not enter cardiomyocytes sufficiently to relieve heart pathology, the efficacy of delivery to various muscles varies greatly, and delivery across the tissue of each skeletal muscle tissue is patchy. PPMOs enter cells at far lower doses, enter cardiomyocytes in useful quantities, and deliver more evenly to myocytes both when different muscles are compared and when assessed at the level of single muscle tissue sections. Compared to PMOs, far lower doses of PPMOs can restore dystrophin sufficiently to reduce disease pathology, increase skeletal and cardiac muscle functions, and prolong survival of animals. The biggest challenge for PPMO is determining safe and effective doses. The toxicity of PPMOs will require caution when moving into the clinic. The first PPMO-based DMD drug is currently in preclinical development for DMD patients who can benefit from skipping exon 50.

Vivo-Morpholino-Based Antiviral for SARS-CoV-2: Implications for Novel Therapies in the Treatment of Acute COVID-19 Disease.

Therapeutic modalities designed specifically to inhibit COVID-19 infection and replication would limit progressive COVID-19-associated pulmonary disease in infected patients and prevent or limit systemic disease. If effective, antivirals could reduce viral transmission rates by reducing viral burden and allow time for immune clearance. For individuals infected with acute-stage disease, antivirals in support of the existing vaccines could reduce COVID-19 hospitalizations and deaths. Here, we evaluate MRCV-19, a phosphorodiamidate morpholino oligo with delivery dendrimer (Vivo-Morpholino), to prevent coronavirus infection in a cell culture model. This is a novel antiviral that effectively inhibits SARS-CoV-2 replication in vitro. By design, MRCV-19 targets the SARS-CoV-2 5'UTR and overlaps the pp1a start site of translation in order to block access of the translation initiation complex to the start. MRCV-19 testing is conducted in a high-throughput, 384-well plate format with a 10-point dose-response curve (common ratio of 2) assayed in duplicate with parallel cytotoxicity evaluations. MRCV-19 was shown to be more effective than hydroxychloroquine and remdesivir in our CPE reduction assay with low toxicity. The clinical translational impact of this study is providing the basis for evaluating MRCV-19 on a large scale in an appropriate infection model for toxicity and systemic high-level inhibition of SARS-CoV-2, which could lead in time to phase I testing in humans.

Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development.

Several vertebrate microRNAs (miRNAs) have been implicated in cellular processes such as muscle differentiation, synapse function, and insulin secretion. In addition, analysis of Dicer null mutants has shown that miRNAs play a role in tissue morphogenesis. Nonetheless, only a few loss-of-function phenotypes for individual miRNAs have been described to date. Here, we introduce a quick and versatile method to interfere with miRNA function during zebrafish embryonic development. Morpholino oligonucleotides targeting the mature miRNA or the miRNA precursor specifically and temporally knock down miRNAs. Morpholinos can block processing of the primary miRNA (pri-miRNA) or the pre-miRNA, and they can inhibit the activity of the mature miRNA. We used this strategy to knock down 13 miRNAs conserved between zebrafish and mammals. For most miRNAs, this does not result in visible defects, but knockdown of miR-375 causes defects in the morphology of the pancreatic islet. Although the islet is still intact at 24 hours postfertilization, in later stages the islet cells become scattered. This phenotype can be recapitulated by independent control morpholinos targeting other sequences in the miR-375 precursor, excluding off-target effects as cause of the phenotype. The aberrant formation of the endocrine pancreas, caused by miR-375 knockdown, is one of the first loss-of-function phenotypes for an individual miRNA in vertebrate development. The miRNA knockdown strategy presented here will be widely used to unravel miRNA function in zebrafish.

Gene knockdowns in adult animals: PPMOs and vivo-morpholinos.

Antisense molecules do not readily cross cell membranes. This has limited the use of antisense to systems where techniques have been worked out to introduce the molecules into cells, such as embryos and cell cultures. Uncharged antisense bearing a group of guanidinium moieties on either a linear peptide or dendrimer scaffold can enter cells by endocytosis and subsequently escape from endosomes into the cytosol/nuclear compartment of cells. These technologies allow systemic administration of antisense, making gene knockdowns and splice modification feasible in adult animals; this review presents examples of such animal studies. Techniques developed with PPMOs, which are an arginine-rich cell-penetrating peptide linked to a Morpholino oligo, can also be performed using commercially available Vivo-Morpholinos, which are eight guanidinium groups on a dendrimeric scaffold linked to a Morpholino oligo. Antisense-based techniques such as blocking translation, modifying pre-mRNA splicing, inhibiting miRNA maturation and inhibiting viral replication can be conveniently applied in adult animals by injecting PPMOs or Vivo-Morpholinos.

A novel L1CAM isoform with angiogenic activity generated by NOVA2-mediated alternative splicing.

The biological players involved in angiogenesis are only partially defined. Here, we report that endothelial cells (ECs) express a novel isoform of the cell-surface adhesion molecule L1CAM, termed L1-ΔTM. The splicing factor NOVA2, which binds directly to L1CAM pre-mRNA, is necessary and sufficient for the skipping of L1CAM transmembrane domain in ECs, leading to the release of soluble L1-ΔTM. The latter exerts high angiogenic function through both autocrine and paracrine activities. Mechanistically, L1-ΔTM-induced angiogenesis requires fibroblast growth factor receptor-1 signaling, implying a crosstalk between the two molecules. NOVA2 and L1-ΔTM are overexpressed in the vasculature of ovarian cancer, where L1-ΔTM levels correlate with tumor vascularization, supporting the involvement of NOVA2-mediated L1-ΔTM production in tumor angiogenesis. Finally, high NOVA2 expression is associated with poor outcome in ovarian cancer patients. Our results point to L1-ΔTM as a novel, EC-derived angiogenic factor which may represent a target for innovative antiangiogenic therapies.

CSGID Solves Structures and Identifies Phenotypes for Five Enzymes in Toxoplasma gondii.

Toxoplasma gondii, an Apicomplexan parasite, causes significant morbidity and mortality, including severe disease in immunocompromised hosts and devastating congenital disease, with no effective treatment for the bradyzoite stage. To address this, we used the Tropical Disease Research database, crystallography, molecular modeling, and antisense to identify and characterize a range of potential therapeutic targets for toxoplasmosis. Phosphoglycerate mutase II (PGMII), nucleoside diphosphate kinase (NDK), ribulose phosphate 3-epimerase (RPE), ribose-5-phosphate isomerase (RPI), and ornithine aminotransferase (OAT) were structurally characterized. Crystallography revealed insights into the overall structure, protein oligomeric states and molecular details of active sites important for ligand recognition. Literature and molecular modeling suggested potential inhibitors and druggability. The targets were further studied with vivoPMO to interrupt enzyme synthesis, identifying the targets as potentially important to parasitic replication and, therefore, of therapeutic interest. Targeted vivoPMO resulted in statistically significant perturbation of parasite replication without concomitant host cell toxicity, consistent with a previous CRISPR/Cas9 screen showing PGM, RPE, and RPI contribute to parasite fitness. PGM, RPE, and RPI have the greatest promise for affecting replication in tachyzoites. These targets are shared between other medically important parasites and may have wider therapeutic potential.

Using Morpholinos to Control Gene Expression.

Morpholino oligonucleotides are stable, uncharged, water-soluble molecules used to block complementary sequences of RNA, preventing processing, read-through, or protein binding at those sites. Morpholinos are typically used to block translation of mRNA and to block splicing of pre-mRNA, though they can block other interactions between biological macromolecules and RNA. Morpholinos are effective, specific, and lack non-antisense effects. They work in any cell that transcribes and translates RNA, but must be delivered into the nuclear/cytosolic compartment to be effective. Morpholinos form stable base pairs with complementary nucleic acid sequences but apparently do not bind to proteins to a significant extent. They are not recognized by any proteins and do not undergo protein-mediated catalysis-nor do they mediate RNA cleavage by RNase H or the RISC complex. This work focuses on techniques and background for using Morpholinos. © 2017 by John Wiley & Sons, Inc.

Making a Morpholino Experiment Work: Controls, Favoring Specificity, Improving Efficacy, Storage, and Dose.

A good Morpholino experiment starts with oligos that have been carefully designed to minimize off-target RNA binding. Performing a successful, reproducible, and well-controlled Morpholino experiment requires oligos that are single stranded and in solution at a known concentration. The outcome of treatment with the oligo needs to be checked for specificity, that is, that the observed outcome is due to interaction with the intended RNA and not an interaction with an unexpected RNA. In this chapter, I will discuss Morpholino use mostly in the context of embryonic microinjection experiments, though many techniques and warnings will be applicable to cell culture or adult animal experiments as well. Controls are critical to a good experiment, but good techniques in designing, preparing, storing, and using the oligos can improve the strength and specificity of the knockdown. Finally, it is important to know the solution concentration of the oligo to ensure that the results are reproducible.

Peptide-assisted delivery of steric-blocking antisense oligomers.

Cationic transport peptides conjugated to steric blocking antisense oligomers (oligos) increase oligo uptake in eukaryotic cell lines, bacteria and mice. Recent reports of arginine-rich transport peptide conjugates strongly suggest that the mechanism of uptake is primarily endocytotic and that previous assay techniques produced confounding artifacts that led to the old non-endocytotic, membrane-penetrating peptide model. The artifacts result from fixing cells for fluorescent microscopy and from using non-trypsinized cells for flow cytometry. Fixing cells redistributes the peptide or peptide-oligo conjugates associated with the outside of cell membranes and trapped in endosomes, giving apparent diffuse cytosolic and nuclear fluorescence. Cationic peptides bound to the outer surface of cells, if not removed, skew fluorescence data obtained by flow cytometry, leading to the earlier conclusions. Upregulation assays now provide a tool for comparing the efficacy of conjugates, measuring oligo uptake by quantitating antisense activity of conjugates. These assays, developed in cell culture and mouse models, are faster and have higher signal-to-noise ratios than downregulation assays. Thus, a convenient and effective method now exists to screen transport peptides.

Revealing details: whole mount microRNA in situ hybridization protocol for zebrafish embryos and adult tissues.

Non-coding microRNA (miRNA) molecules bind their target mRNAs and thereby modulate the amount of protein produced. To understand the significance of a potential miRNA-mRNA interaction, temporal and spatial information on miRNA and mRNA expression is essential. Here, we provide a detailed protocol for miRNA whole mount in situ hybridization. We introduce the use of Morpholino based oligos as antisense probes for miRNA detection, in addition to the current "gold standard" locked nucleic acid (LNA) probes. Furthermore we have modified existing miRNA in situ protocols thereby improving both sensitivity and resolution of miRNA visualization in whole zebrafish embryos and adult tissues.

Using Morpholinos to control gene expression.

Morpholino oligonucleotides are stable, uncharged, water-soluble molecules that bind to complementary sequences of RNA, thereby inhibiting mRNA processing, read-through, and protein binding at those sites. Morpholinos are typically used to inhibit translation of mRNA, splicing of pre-mRNA, and maturation of miRNA, although they can also inhibit other interactions between biological macromolecules and RNA. Morpholinos are effective, specific, and lack non-antisense effects. They work in any cell that transcribes and translates RNA. However, unmodified Morpholinos do not pass well through plasma membranes and must therefore be delivered into the nuclear or cytosolic compartment to be effective. Morpholinos form stable base pairs with complementary nucleic acid sequences but apparently do not bind to proteins to a significant extent. They are not recognized by proteins and do not undergo protein-mediated catalysis; nor do they mediate RNA cleavage by RNase H or the RISC complex. This work focuses on techniques and background for using Morpholinos.

Using morpholinos to control gene expression.

Morpholino oligonucleotides are stable, uncharged, water-soluble molecules used to block complementary sequences of RNA, preventing processing, read-through, or protein binding at those sites. Morpholinos are typically used to block translation of mRNA and to block splicing of pre-mRNA, though they can block other interactions between biological macromolecules and RNA. Morpholinos are effective, specific, and lack non-antisense effects. They work in any cell that transcribes and translates RNA, but must be delivered into the nuclear/cytosolic compartment to be effective. Morpholinos form stable base pairs with complementary nucleic acid sequences but apparently do not bind to proteins to a significant extent. They are not recognized by any proteins and do not undergo protein-mediated catalysis; nor do they mediate RNA cleavage by RNase H or the RISC complex. This work focuses on techniques and background for using Morpholinos.